The present invention relates to the use of primers in polymerase chain reaction assays for the detection of fungal pathogens in wheat and barley. The use of these primers enables the detection of specific isolates of fungal pathogens and the monitoring of disease development in plant populations.
Diseases in plants cause considerable crop loss from year to year resulting both in economic deprivation to farmers and, in many parts of the world, to shortfalls in the nutritional provision for local populations. The widespread use of fungicides has provided considerable security against plant pathogen attack. However, despite $1 billion worth of expenditure on fungicides, worldwide crop losses amounted to approximately 10% of crop value in 1981 (James, 1981; Seed Sci. and Technol. 9: 679-685).
The severity of the destructive process of disease depends on the aggressiveness of the pathogen and the response of the host. One aim of most plant breeding programs is to increase the resistance of host plants to disease. Typically, different races of pathogens interact with different varieties of the same crop species differentially, and many sources of host resistance only protect against specific pathogen races. Furthermore, some pathogen races show early signs of disease symptoms, but cause little damage to the crop. Jones and Clifford (1983; Cereal Diseases, John Wiley) report that virulent forms of the pathogen are expected to emerge in the pathogen population in response to the introduction of resistance into host cultivars and that it is therefore necessary to monitor pathogen populations. In addition, there are several documented cases of the evolution of fungal strains that are resistant to particular fungicides. As early as 1981, Fletcher and Wolfe (1981; Proc. 1981 Brit. Crop Prot. Conf.) contended that 24% of the powdery mildew populations from spring barley and 53% from winter barley showed considerable variation in response to the fungicide triadimenol and that the distribution of these populations varied between varieties, with the most susceptible variety also giving the highest incidence of less susceptible types. Similar variation in the sensitivity of fungi to fungicides has been documented for wheat mildew (also to triadimenol), Botrytis (to benomyl), Pyrenophora (to organomercury), Pseudocercosporella (to MBC-type fungicides) and Mycosphaerella fijiensis to triazoles to mention just a few (Jones and Clifford; Cereal Diseases, John Wiley, 1983).
Wheat is currently the most important agricultural commodity in international markets and occupies about 20% of the world""s farmed land (1977; Compendium of Wheat Diseases, Amer. Phytopath. Soc. page 1). Eightly percent of the world""s supply of wheat is grown in North America, Europe, China, and the Soviet Union. Approximately 20% of the worldwide production of wheat is lost to disease annually.
Pyrenophora tritici-repentis (Died.) Drechs. (syn. P. trichostoma (Fr.) Fckl.), anamorph Drechslera tritici-repentis (Died.) Shoem. (syn. Helminthosporium tritici-repentis Died.), causes tan spot also known as yellow spot of wheat worldwide (1977; Compendium of Wheat Diseases, Amer. Phytopath. Soc. page 42). It has resulted in wheat yield losses from 3 to 50 % in Australia, South America, and North America and has been recently considered the most important foliar wheat disease in North Dakota (Zhang et al., 1997; Phytopathology. Vol.87:154-160). It can also contribute to leaf-spotting complexes with other foliar pathogens. Current disease control measures include fungicide application and cultural practices including destroying wheat stubble, using pathogen-free seed and wide row plant spacing to reduce foliage density and relative humidity in the wheat canopy.
Pyrenophora teres Drechs., anamorph Drechslera teres (Sacc.) Shoem. (syn. Helminthosporium teres Sacc.) causes net blotch primarily in barley; however, sporadic infections also occur in wheat (Jones and Clifford; Cereal Diseases, John Wiley, 1983). Typical yield losses due to net blotch are between 10 to 40%. Yield losses can approach 100% in fields containing susceptible cultivars (1982; Compendium of Barley Diseases, Amer. Phytopath. Soc. page 22). In addition to affecting overall grain yield and weight, the disease also reduces the carbohydrate content. This reduces malt extract yield and therefore lowers the brewing quality of the grain.
In view of the above, there is a real need for the development of technology that will allow the identification of specific races of pathogen fungi early in the infection process. By identifying the specific race of a pathogen before disease symptoms become evident in the crop stand, the agriculturist can assess the likely effects of further development of the pathogen in the crop variety in which it has been identified and can choose an appropriate fungicide if such application is deemed necessary.
In view of the above, a primary object of the invention is to provide a method for the identification of specific races of pathogen fungi early in the infection process. The invention therefore provides Internal Transcribed Spacer (ITS) DNA sequences that show variability between different fungal pathotypes. Such DNA sequences are useful in the method of the invention as they can be used to derive primers for use in polymerase chain reaction (PCR)-based diagnostic assays. These primers generate unique fragments in PCR reactions in which the DNA template is provided by specific fungal pathotypes and can thus be used to identify the presence or absence of specific pathotypes in host plant material before the onset of disease symptoms.
In a preferred embodiment, the invention provides novel ITS1 and ITS2 DNA sequences for the fungal pathogen Pyrenophora tritici-repentis. In another preferred embodiment, the invention provides ITS-derived diagnostic primers for the detection of Pyrenophora tritici-repentis. In an additional preferred embodiment, the invention provides novel ITS-derived diagnostic primers that are useful for the detection of not only Pyrenophora tritici-repentis, but also, surprisingly, Pyrenophora teres and Drechslera sorokiniana. The present invention therefore addresses a long-felt but unfulfilled need to identify different pathotypes of plant pathogenic fungi, especially those that cause tan spot in wheat.
This invention provides the possibility of assessing potential damage in a specific crop variety-pathogen strain relationship and of utilizing judiciously the diverse armory of fungicides that is available. Furthermore, the invention can be used to provide detailed information on the development and spread of specific pathogen races over extended geographical areas. The invention provides a method of detection that is especially suitable for diseases with a long latent phase.
Kits useful in the practice of the invention are also provided. The kits find particular use in the identification of the fungal pathogen Pyrenophora tritici-repentis. 
SEQ ID NO:1 Oligonucleotide Primer ITS1.
SEQ ID NO:2 Oligonucleotide Primer ITS2.
SEQ ID NO:3 Oligonucleotide Primer ITS3.
SEQ ID NO:4 Oligonucleotide Primer ITS4.
SEQ ID NO:5 M13 Universal-20 Primer.
SEQ ID NO:6 Reverse Primer used in Example 2.
SEQ ID NO:7 Oligonucleotide Primer JB629.
SEQ ID NO:8 Oligonucleotide Primer JB630.
SEQ ID NO:9 Oligonucleotide Primer JB631.
SEQ ID NO:10 Oligonucleotide Primer JB632.
SEQ ID NO:11 Oligonucleotide Primer JB633.
SEQ ID NO:12 Oligonucleotide Primer JB634.
SEQ ID NO:13 Oligonucleotide Primer JB635.
SEQ ID NO:14 Oligonucleotide Primer JB636.
SEQ ID NO:15 Oligonucleotide Primer JB637.
SEQ ID NO:16 Oligonucleotide Primer JB638.
SEQ ID NO:17 Oligonucleotide Primer JB651.
SEQ ID NO:18 Oligonucleotide Primer JB652.
SEQ ID NO:19 Oligonucleotide Primer JB653.
SEQ ID NO:20 Oligonucleotide Primer JB654.
SEQ ID NO:21 Oligonucleotide Primer JB655.
SEQ ID NO:22 Oligonucleotide Primer JB656.
SEQ ID NO:23 Oligonucleotide Primer JB657.
SEQ ID NO:24 Oligonucleotide Primer JB658.
SEQ ID NO:25 Oligonucleotide Primer JB659.
SEQ ID NO:26 Oligonucleotide Primer JB660.
SEQ ID NO:27 Oligonucleotide Primer JB675.
SEQ ID NO:28 Oligonucleotide Primer JB676.
SEQ ID NO:29 Consensus DNA sequence of the ITS region from Pyrenophora tritici-repentis isolates 6715, 119-2-3, DL22, PTR4A, 44184, 205, 403, 109, 407, 1316, and 223, comprising in the 5xe2x80x2 to 3xe2x80x2 direction: 3xe2x80x2 end of the small subunit rRNA gene, Internal Transcribed Spacer 1, 5.8S rRNA gene, Internal Transcribed Spacer 2, and 5xe2x80x2 end of the large subunit rRNA gene.
SEQ ID NO:30 Consensus DNA sequence of the ITS region from Pyrenophora tritici-repentis isolate 6715 clones 2 and 4, comprising in the 5xe2x80x2 to 3xe2x80x2 direction: 3xe2x80x2 end of the small subunit rRNA gene, Internal Transcribed Spacer 1, 5.8S rRNA gene, Internal Transcribed Spacer 2, and 5xe2x80x2 end of the large subunit rRNA gene.
SEQ ID NO:31 DNA sequence of the ITS region from Pyrenophora tritici-repentis isolate 119-2-3 clone 2-2, comprising in the 5xe2x80x2 to 3xe2x80x2 direction: 3xe2x80x2 end of the small subunit rRNA gene, Internal Transcribed Spacer 1, 5.8S rRNA gene, Internal Transcribed Spacer 2, and 5xe2x80x2 end of the large subunit rRNA gene.
SEQ ID NO:32 DNA sequence of the ITS region from Pyrenophora tritici-repentis isolate DL22 clone 1-1, comprising in the 5xe2x80x2 to 3xe2x80x2 direction: 3xe2x80x2 end of the small subunit rRNA gene, Internal Transcribed Spacer 1, 5.8S rRNA gene, Internal Transcribed Spacer 2, and 5xe2x80x2 end of the large subunit rRNA gene.
SEQ ID NO:33 DNA sequence of the ITS region from Pyrenophora tritici-repentis isolate PTR4A clone 2-3, comprising in the 5xe2x80x2 to 3xe2x80x2 direction: 3xe2x80x2 end of the small subunit rRNA gene, Internal Transcribed Spacer 1, 5.8S rRNA gene, Internal Transcribed Spacer 2, and 5xe2x80x2 end of the large subunit rRNA gene.
SEQ ID NO:34 DNA sequence of the ITS region from Pyrenophora tritici-repentis isolate 44184 clone 3-1, comprising in the 5xe2x80x2to 3xe2x80x2 direction: 3xe2x80x2 end of the small subunit rRNA gene, Internal Transcribed Spacer 1, 5.8S rRNA gene, Internal Transcribed Spacer 2, and 5xe2x80x2 end of the large subunit rRNA gene.
SEQ ID NO:35 DNA sequence of the ITS region from Pyrenophora tritici-repentis isolate 205 clone 4-2, comprising in the 5xe2x80x2 to 3xe2x80x2 direction: 3xe2x80x2 end of the small subunit rRNA gene, Internal Transcribed Spacer 1, 5.8S rRNA gene, Internal Transcribed Spacer 2, and 5xe2x80x2 end of the large subunit rRNA gene.
SEQ ID NO:36 DNA sequence of the ITS region from Pyrenophora tritici-repentis isolate 403 clone 5-2, comprising in the 5xe2x80x2 to 3xe2x80x2 direction: 3xe2x80x2 end of the small subunit rRNA gene, Internal Transcribed Spacer 1, 5.8S rRNA gene, Internal Transcribed Spacer 2, and 5xe2x80x2 end of the large subunit rRNA gene.
SEQ ID NO:37 DNA sequence of the ITS region from Pyrenophora tritici-repentis isolate 109 clone 6-2, comprising in the 5xe2x80x2 to 3xe2x80x2 direction: 3xe2x80x2 end of the small subunit rRNA gene, Internal Transcribed Spacer 1, 5.8S rRNA gene, Internal Transcribed Spacer 2, and 5xe2x80x2 end of the large subunit rRNA gene.
SEQ ID NO:38 DNA sequence of the ITS region from Pyrenophora tritici-repentis isolate 407 clone 7-3-2, comprising in the .5xe2x80x2 to 3xe2x80x2 direction: 3xe2x80x2 end of the small subunit rRNA gene, Internal Transcribed Spacer 1, 5.8S rRNA gene, Internal Transcribed Spacer 2, and 5xe2x80x2 end of the large subunit rRNA gene.
SEQ ID NO:39 DNA sequence of the ITS region from Pyrenophora tritici-repentis isolate 1316 clone 8-1, comprising in the 5xe2x80x2 to 3xe2x80x2 direction: 3xe2x80x2 end of the small subunit rRNA gene, Internal Transcribed Spacer 1, 5.8S rRNA gene, Internal Transcribed Spacer 2, and 5xe2x80x2 end of the large subunit rRNA gene.
SEQ ID NO:40 DNA sequence of the ITS region from Pyrenophora tritici-repentis isolate 223 clone 9-2, comprising in the 5xe2x80x2 to 3xe2x80x2 direction: 3xe2x80x2 end of the small subunit rRNA gene, Internal Transcribed Spacer 1, 5.8S rRNA gene, Internal Transcribed Spacer 2, and 5xe2x80x2 end of the large subunit rRNA gene.
SEQ ID NO:41 DNA sequence of the ITS region from Pyrenophora teres isolate 36570 clone 10-1, comprising in the 5xe2x80x2 to 3xe2x80x2 direction: 3xe2x80x2 end of the small subunit rRNA gene, Internal Transcribed Spacer 1, 5.8S rRNA gene, Internal Transcribed Spacer 2, and 5xe2x80x2 end of the large subunit rRNA gene.
The present invention provides unique DNA sequences that are useful in identifying different pathotypes of plant pathogenic fungi. Particularly, the DNA sequences can be used as primers in PCR-based analysis for the identification of fungal pathotypes. The DNA sequences of the invention include the Internal Transcribed Spacer (ITS) sequences of the ribosomal RNA gene regions of particular fungal pathogens as well as primers derived from these regions that are capable of identifying the particular pathogen. These ITS DNA sequences from different pathotypes within a pathogen species or genus, which vary between the different members of the species or genus, can be used to identify those specific members.
Biomedical researchers have used PCR-based techniques for some time and with moderate success to detect pathogens in infected animal tissues. Only recently, however, has this technique been applied to detect plant pathogens. The presence of Gaumannomyces graminis in infected wheat has been detected using PCR of sequences specific to the pathogen mitochondrial genome (Schlesser et al., 1991; Applied and Environ. Microbiol 57: 553-556), and random amplified polymorphic DNA (i.e. RAPD) markers were able to distinguish numerous races of Gremmeniella abietina, the causal agent of scleroderris canker in conifers. U.S. Pat. No. 5,585,238 (herein incorporated by reference in its entirety) describes primers derived from the ITS sequences of the ribosomal RNA gene region of strains of Septoria, Pseudocercosporella, and Mycosphaerella and their use in the identification of these fungal isolates using PCR-based techniques. In addition, U.S. patent application Ser. No. 08/722,187 (herein incorporated by reference in its entirety) describes primers derived from the ITS sequences of the ribosomal RNA gene region of strains of Fusarium and their use in the identification of these fungal isolates using PCR-based techniques. Furthermore, U.S. patent application Ser. No. 08/742,023 (herein incorporated by reference in its entirety) describes primers derived from the ITS sequences of the ribosomal RNA gene region of strains of Cercospora, Helminthosporium, Kabatiella, and Puccinia and their use in the identification of these fungal isolates using PCR-based techniques.
Ribosomal genes are suitable for use as molecular probe targets because of their high copy number. Despite the high conservation between mature rRNA sequences, the non-transcribed and transcribed spacer sequences are usually poorly conserved and are thus suitable as target sequences for the detection of recent evolutionary divergence. Fungal rRNA genes are organized in units, each of which encodes three mature subunits of 18S (small subunit), 5.8S, and 28S (large subunit). These subunits are separated by two Internal Transcribed Spacers, ITS 1 and ITS2, of around 300 bp (White et al., 1990; In: PCR Protocols; Eds.: Innes et al.; pages 315-322). In addition, the transcriptional units are separated by non-transcribed spacer sequences (NTSs). The ITS and NTS sequences are particularly suitable for the detection of specific pathotypes of different fungal pathogens.
The DNA sequences of the invention are from the Internal Transcribed Spacer sequences of the ribosomal RNA gene region of different plant pathogens. The ITS DNA sequences from different pathotypes within a pathogen species or genus vary among the different members of the species or genus. After determining the ITS sequences of a pathogen, these sequences can be aligned with other ITS sequences. Primers can then be derived from the ITS sequences. That is, primers can be designed based on regions within the ITS sequences having the greatest differences in sequence among the fungal pathotypes. The sequences and primers based thereon can be used to identify specific pathogens.
Particular DNA sequences of interest include ITS DNA sequences from Pyrenophora tritici-repentis and Pyrenophora teres. Such ITS DNA sequences are disclosed in SEQ ID NOs: 29-41. Sequences of representative oligonucleotide primers derived from these ITS sequences are disclosed in SEQ ID NOs: 7-28. The sequences find use in the PCR-based identification of the pathogens of interest. Therefore, based on Applicants"" disclosure of the present invention, the fungal pathogen responsible for tan spot in wheat can, for the first time, be detected by a PCR-based diagnostic assay.
Methods for the use of the primer sequences of the invention in PCR analysis are well known in the art. For example, see U.S. Pat. Nos. 4,683,195 and 4,683,202, as well as Schlesser et al. (1991) Applied and Environ. Microbiol 57:553-556. See also, Nazar et al. (1991; Physiol and Molec. Plant Pathol. 39: 1-11), which used PCR amplification to exploit differences in the ITS regions of Verticillium albo-atrum and Verticillium dahliae and therefore distinguish between the two species; and Johanson and Jeger (1993; Mycol. Res. 97: 670-674), who used similar techniques to distinguish the banana pathogens Mycosphaerella fijiensis and Mycospharella musicola. 
The ITS DNA sequences of the invention can be cloned from fungal pathogens by methods known in the art. In general, the methods for the isolation of DNA from fungal isolates are known. See, Raeder and Broda (1985) Letters in Applied Microbiology 2:17-20; Lee et al. (1990) Fungal Genetics Newsletter 35:23-24; and Lee and Taylor (1990) In: PCR Protocols: A Guide to Methods and Applications, Innes et al. (Eds.); pages 282-287.
Alternatively, the ITS sequences of interest can be determined by PCR amplification. In an exemplified embodiment, primers to amplify the entire ITS region were designed according to White et al. (1990; In: PCR Protocols; Eds.: Innes et al. pages 315-322), and the amplified ITS sequence was subcloned into the pCRII cloning vector. The subcloned sequence included the left hand ITS (ITS 1), the right hand ITS (ITS2), as well as the centrally located 5.8S rRNA gene. This was undertaken for several isolates of Pyrenophora tritici-repentis and from a single isolate of Pyrenophora teres. 
The determined ITS sequences were compared within each pathogen group to locate divergences that might be useful to test in PCR to distinguish the different species and/or strains. The ITS DNA sequences that were determined are shown in SEQ ID NOs: 29-41. From the identification of divergences, numerous primers were synthesized and tested in PCR-amplification. Templates used for PCR-amplification testing were firstly purified pathogen DNA, and subsequently DNA isolated from infected host plant tissue. Thus, it was possible to identify pairs of primers that were diagnostic, i.e. that identified one particular pathogen species or strain but not another species or strain of the same pathogen. Primers were also designed to regions highly conserved among the species to develop genus-specific primers as well as primers that identify any of several fungal pathogens that cause a certain disease. For example, primers were developed to detect both P. teres and P. tritici-repentis. 
Preferred primer combinations are able to distinguish between the different species or strains in infected host tissue, i.e. host tissue that has previously been infected with a specific pathogen species or strain. This invention provides numerous primer combinations that fulfill this criterion for P. teres and P. tritici-repentis. The primers of the invention are designed based on sequence differences among the fungal ITS regions. A minimum of one base pair difference between sequences can permit design of a discriminatory primer. Primers designed to a specific fungal DNA""s ITS region can be used in combination with a primer made to a conserved sequence region within the ribosomal DNA""s coding region to amplify species-specific PCR fragments. In general, primers should have a theoretical melting temperature between about 60 to about 70xc2x0 C. to achieve good sensitivity and should be void of significant secondary structure and 3xe2x80x2 overlaps between primer combinations. Primers generally have sequence identity with at least about 5-10 contiguous nucleotide bases of ITS1 or ITS2. In preferred embodiments, primers are anywhere from about 5-30 nucleotide bases long and are preferably at least 10 nucleotide bases long.
As an alternative to the above-described PCR diagnostic techniques, a specific fungal DNA""s ITS region can be used to design polynucleotide label probes, each comprising a label portion and a nucleic acid region having a sequence that is complementary to at least about 5-10 contiguous nucleotides of ITS 1 or ITS2. In preferred embodiments, the complementary nucleic acid regions of such probes are anywhere from about 5-30 nucleotide bases long and are preferably at least 10 nucleotide bases long. The label portion of the probes may comprise either a label that provides a signal or a binding site for such a label. Polynucleotide label probes such as these may be used in fungal detection methods that involve the following steps: (a) designing at least one polynucleotide label probe comprising a nucleic acid region having a sequence that is complementary to at least about 5-10 contiguous nucleotide bases of ITS1 or ITS2 of a fungal pathogen, such as Pyrenophora tritici-repentis, and a label portion comprising either a label that provides a signal when said probe hybridizes with another nucleic acid molecule or a binding site for said label; (b) isolating DNA from plant tissue infected with the fungal pathogen; (c) hybridizing the DNA of step (b) with the probe or probes of step (a); and (d) detecting the fungal pathogen by detecting the label, wherein detection of the label indicates that the hybridization of step (c) has occurred and the fungal pathogen is present. Labels may be designed and detected according to any means known in the art, such as through the use of radioisotopes, fluorescence, or a planar optical waveguide. See, for example, U.S. Pat. No. 4,868,105, WO 95/33197, and WO 95/33198, all of which are incorporated herein by reference.
The present invention lends itself readily to the preparation of xe2x80x9ckitsxe2x80x9d containing the elements necessary to carry out the process. Such a kit may comprise a carrier being compartmentalized to receive in close confinement therein one or more containers, such as tubes or vials. One of the containers may contain unlabeled or detectably labeled DNA primers or probes, which may be present in lyophilized form or in an appropriate buffer as necessary. One or more containers may contain one or more enzymes or reagents to be utilized in PCR reactions. These enzymes may be present by themselves or in admixtures, in lyophilized form or in appropriate buffers. Finally, the kit may contain all of the additional elements necessary to carry out the technique of the invention, such as buffers, extraction reagents, enzymes, pipettes, plates, nucleic acids, nucleoside triphosphates, filter paper, gel materials, transfer materials, autoradiography supplies, and the like.
The examples below show typical experimental protocols that can be used for isolating ITS sequences, selecting suitable primer sequences, testing primers for selective and diagnostic efficacy, and using such primers for disease and fungal isolate detection. The examples are provided by way of illustration and not by way of limitation.